1. Field of the Invention
The present invention relates to methods and reagents to detect viral DNA in atypical Pap smears indicative of predisposition for cancer.
2. Background of the Invention
Human papilloma virus (HPV) is one of the most common sexually transmitted viral diseases, being manifested in over one million people per year in the United States, and the primary etiologic agent for cervical cancer in women worldwide. There are more than 100 distinct types of HPV, of which approximately 50 are found in the lower genital tract. Of the HPV types found in genital lesions, about 15-20 have been implicated in cervical precancers and cancers. The HPV viral types associated with cervical cancer can be ranked as high-risk, intermediate-risk, or low-risk, according to the tendency of infected individuals to progress to cancer. Common high-risk types include: 16, 18, 39, 45, and 56; intermediate-risk types include: 31, 33, 35, 51, 52, and 58; and low-risk types include: 6, 11, 42, 43, and 44.
HPV is a DNA virus having a genome of about 7900 base pairs in length that contains seven open reading frames that encode five proteins that are expressed early in infection and two that are expressed late in infection. Proteins E6 and E7 are linked to the induction of transformation of benign cells in vitro and in vivo. While the exact mechanism has not yet been established, one of the proteins from high-risk strains appears to inactivate the human tumor suppressor gene p53.
Traditionally, the Papanicolaou (Pap) smear assay has been used to screen individuals for HPV-associated cervical lesions, with Pap smears being performed on women annually to check for the presence of atypical or cancerous cells. Roughly 90% of all smears are normal, 3% are unequivocally dysplastic including LSIL (low-grade squamous intraepithelial lesion), and 7% are squamous atypias (ASC-US; atypical cells of uncertain significance). Patients with a diagnosis of dysplasia are brought back to their doctor's office for biopsy and further procedures. The ASC-US and LSIL diagnoses present the doctor and patient with multiple choices for treatment. If these patients could be tested for the presence of low-risk and high-risk HPV types, such information would greatly aid in determining an optimal course of treatment. For example, the presence of a low-risk HPV type could indicate that no further action—except perhaps for more frequent Pap smears—was required. On the other hand, the presence of a high-risk HPV type could indicate the need for a more aggressive approach, including a cone biopsy, loop electrode excision procedure (LEEP), or ablative therapy (laser surgery or cryotherapy).
An alternative to standard Pap smears is the ThinPrep® Pap test (Cytyc Corp.). A standard Pap smear involves sampling the uterine cervix with a spatula or cytobrush and smearing the cells directly onto a glass slide. The ThinPrep® Pap test involves suspending the sample in a buffered fixative solution which provides better sample recovery, fewer artifacts, multiple slides from one sample, less cell crowding, overlap, and better cellular morphology. In a ThinPrep® Pap test sample, the sampling device is rinsed into a buffered preservative solution and this solution is used to make slides. By suspending the sample in preservative solution and using the ThinPrep® machine to make the slides, the resulting slide is a thin-layer preparation that is clearer of blood, inflammatory cells, mucus, and other obscuring artifacts than a standard Pap smear. The FDA has found the ThinPrep® Pap test to be preferable to the standard Pap smear for the detection of LSIL and more severe lesions. Another advantage of the ThinPrep® Pap test is that the entire sample is not used to make a single slide, and therefore, preserved cells from the unused portion of the sample can be used to make additional slides for the purpose of performing HPV testing.
Both of the standard methods for detecting cervical cancer (i.e., the Pap smear and ThinPrep® Pap test) can be used to detect atypical or cancerous cells, but not HPV infection. Pap smears are classified as: normal; ASC-US (atypical cells of uncertain significance); LSIL (low-grade squamous intraepithelial lesion); or HSIL (high-grade squamous intraepithelial lesion). Currently, about 4.4 million Pap smears are classified as ASC-US and 2.5 million Pap smears are classified as LSIL, annually. This indicates that 6.9 million or 7-8% of all Pap smears per year are ambiguous in their results. Knowledge of the particular HPV type present would aid the patient and clinician in determining the level of risk and treatment options.
Patients with an ambiguous cytology may still have preinvasive or microinvasive cancer and HPV DNA typing may aid in differentiating patients. Studies have shown that ambiguous cytology and high-risk HPV infection with types 16, 18, and 31 are more likely to have high grade SIL or microinvasive histopathology on biopsy. Studies have also suggested that acute infection with HPV types 16 and 18 confer an 11 to 16.9 fold risk of rapid development of cervical intraepithelial neoplasia (CIN).
Accordingly, a need exists to differentiate the borderline Pap smears and ThinPrep® Pap tests based on the HPV type present, if any. Other attempts at preparing oligonucleotide probes have been reported. See U.S. Pat. Nos. 4,820,530; 4,849,311; 4,849,332; 4,849,334; 4,908,306; 4,983,728; 5,057,411; 5,411,847; 5,484,699; 5,501,947; 5,527,898; 5,554,538; and 5,679,509; European Patent No. 0 477 972 B1; and Cole et al., 1987, J. Mol. Biol. 193:599-608. Applicants are also aware of U.S. Pat. No. 5,538,871. However, each oligonucleotide probe appears to be specific to only one HPV type and, therefore, numerous probes would be needed to form a complete set capable of detecting all high-risk HPV types.
All patents and references cited herein are explicitly incorporated by reference in their entirety.